Chromatography media for biomolecules have traditionally been categorised according to one or more of the following possible modes of interaction with the sample:                Hydrophobic Interaction (<<reversed phase>>)        Hydrophilic Interaction (<<normal phase>>)        Cation Exchange        Anion Exchange        Size Exclusion        Metal Ion Chelation        
Perpetual improvements in the titres of technical fermentation processes led to an increased demand of simple, cost-effective, and highly selective downstream purification technologies capable of handling large protein capacities without up-scaling the required volumes of liquid by the same factor. Traditional stepwise application of the above chromatographic categories to a given separation problem was accordingly mirrored in a step-by-step, steady improvement of the product purity but also in product losses at every stage which accumulate seriously in the end, not to mention the operational time and cost of goods. Introduction of affinity chromatography at an early stage into the downstream process could be an answer to this demand since the reduction of a consecutive series of sequential chromatography steps into only one could thus be demonstrated many times. Affinity chromatography is sometimes regarded as a class of its own although, from a chemical point of view, it is based on the same interaction modes as above, but usually on a combination of two or more modes. The principal characteristic of affinity chromatography is its high specificity of a pre-determined analyte which is usually based on a known molecular recognition pair of biological significance such as antigen-antibody, carbohydrate-lectin, hormone-receptor, or between complementary nucleic acid strands. Most affinity sorbents are therefore made-to-measure by the end-user according to his particular separation task. To yield a fully functional sorbent, the biological affinity residue is coupled—immediately or via an optional tether allowing more degrees of freedom in the translational and rotational motion of the residue—by a choice of only a few standard bioconjugation techniques to a support material which itself may be commercially available. The shelf-life of such a sorbent is normally only short, and it has often to be prepared on-demand.
Additionally, synthetic affinity ligands such as short linear or cyclic synthetic peptides or peptidomimetics, but also certain reactive dyes (mainly triazine dyes) have been found to interact group-specifically with biomolecules. The latter are inexpensive and easy-to-prepare low-molecular weight residues which lack the disadvantages of the labilities and variabilities in the tertiary structures of biopolymers. Moreover, due to their small molecular sizes and tunable, robust activation chemistries, they can be efficiently immobilised in a directed orientation onto solid supports even without long tethering, whereas biopolymers under the same conditions often suffer from lack of activity after immobilisation due to defolding, steric hindrance, or random orientation. In either case, the component of the sorbent which is actively involved in the recognition process is usually only present on the surface (often as a surface-bound monolayer) of a supporting solid.
Apart from homogeneous solid support materials, sorbents consisting of a 2-layered cross-sectional morphology according to the general scheme of a bulk solid support material whose surface is covered with a thin film of a crosslinked polymer are well-known from the state of the art. Polymers such as heavily (radiation-)crosslinked polybutadiene, polystyrene, polysiloxane, poly(meth)acrylate, and polyamides have primarily been used in the past. They have been employed primarily with the intent of creating a dense interface which shields the surrounding medium from unwanted interactions with the underlying part (“carrier”) of the solid support material. Such interactions may lead to unspecific or even irreversible binding of biomolecules to the sorbent while, on the other hand, constituents of the solid support material or its chemical linkages to the residues may be corroded by aggressive components of either the sample or the eluent. Polymer-coated sorbents are basically known for applications in all chromatographic categories as they are listed above, but in particular for hydrophobic interaction and size exclusion. Also known are polymer coatings which are not internally crosslinked but grafted to the carrier material as linear or branched chains, such as the so-called tentacle resins.
Affinity chromatography, on the other hand, has mostly been carried out with bulk gel-phase resins. Pre-eminent gel-forming materials are medium-crosslinked polysaccharides, polyacrylamides, and poly(ethylene oxides). Such hydrogels ensure a biocompatible interface which can well accommodate both the active residue and the biological analyte interacting therewith due to their softness (conformational flexibility, elastic modulus), large pore systems, high polarity and high water content, as well as the absence of reactive or denaturing chemical groups. They are able to retain proteins in their native state, i.e. preserve their correctly folded, three-dimensional structure, state of association, and functional integrity. This is to a large part a consequence of the fact that organic solvents which are often required to elute proteins or peptides from strongly adsorbing, hydrophobic (<<hard>>) media, can be avoided. Lack of intrinsic adsorption strength of the support is thereby compensated by the introduction of highly-specific, intact biological ligands as binding partners for the separation target which are well accommodated within the hydrogel. The mechanical resistance of these media is, however, much weaker than that of inorganic support materials since they are compressible under an applied pressure and do not tolerate shear stress caused by agitation, column packing or high liquid flow rates. Affinity sorbents that are fully compatible with robust HPLC process conditions are therefore rare.
Only in the recent past it has been recognised that the mechanical resistance of the stationary phase is a bulk property of the sorbent support whereas only a thin layer at the interface between the stationary and the mobile phases is responsible for mass exchange and for the interaction with the biological analyte. Therefore the concept of combining the function of a mechanically very rigid and dimensionally stable, porous 3-dimensional core, and a biocompatible, gel-like interface layer which carries the active residues for binding the analyte has been brought up, and the associated synthetic problems have been technically solved. Such hybrid materials employ loosely crosslinked polymers of high polarity on a base of either an inorganic oxide or a densely crosslinked polymer of low polarity.
Methodologically, they can be prepared by applying the polymer of high polarity onto the core material or by directly polymerising polar monomers, precursors thereof or a prepolymer in the presence of the core material and a crosslinker. The majority of materials prepared according to the latter method is being described in the literature as having either a non-pore-penetrating or a pore-filling morphology. While non-penetrating films suffer from restricted surface areas available for interaction with the analyte and thus low binding capacities which only depend on the thickness of the polymer film, pore filling films take advantage of the full inner pore volume of the core material in the interaction with an analyte, which usually results in good binding capacities but slow diffusional mass transfer rates inside the pores and exchange kinetics with the mobile phase. A polymer film covering, but not filling completely, the interior surfaces of the core material, would be beneficial in this respect. The best known representative of this whole class of sorbents is the system which consists of branched and optionally further crosslinked polyethylene imine grafted onto a porous silica support core material. It has been demonstrated that such sorbents can be further derivatised but they have been commercialised only for ion exchange and those group-specific affinity applications which require only small standard residues.
A conceptually different approach to the production of synthetic affinity media is the so-called <<molecular imprinting>> technique which is based on shape and functional group complementarity between the target substrate and polymeric cavities formed during a polymerisation reaction which is carried out in the presence of the target substrate and a porogen, which have to be removed subsequently. Imprinting has been developed for a large number of substrates including proteins and peptides, and can be split in a covalent and a non-covalent method, as far as the temporary fixation of the target is concerned. It is, however, restricted to the formation of a few highly-crosslinked types of polymers as solid support materials and has so far not found widespread acceptance once the production scale is reached, especially not for pharmaceutical proteins or peptides which are under the control of a regulatory body.
The most widespread used affinity media for the purification of immunoglobulins G (IgG) are support-bound proteins A or G, both of which are naturally produced on the cell walls of Staphylococci, as well as protein L, but all require rather high capital investments for large-scale applications, which basically prevent their use as disposables. Protein A is known to bind a particular epitope on the constant Fc part of antibodies. It is therefore of limited use in the purification of recombinant antibody fragments or fusion products lacking this region. Repeated use of protein-derived sorbents is, on the other hand, associated with the disadvantages of protein secondary/tertiary structure and/or chemical linkage instability towards harsh manufacturing conditions, resulting in possible inactivation or leakages especially during obligatory, strongly alkaline sanitisation treatments in between chromatographic runs. In addition to an accordingly reduced life-span there is an ongoing debate as to the application of protein A sorbents in pharmaceutical production since even minute amounts of leaked protein A are suspected to cause immunological disorders in humans when products to be purified are for in vivo pharmaceutical use. Thus, registration approval and expected market authorisation for a regulated product are other important factors in the decision for a technical purification process, and therefore it has become an industry standard that protein A chromatography must be followed by an additional chromatography step in order to remove leached toxicants.
Beside attempts of creating engineered variants of these proteins with improved technical properties, as a consequence also a few sorbents having either very short (unnatural) peptide epitopes only or even fully synthetic residues were manufactured. Those synthetic media useful as protein A/G/L alternatives which are commercially available have recently been reviewed in the January 2007 issue of Journal of Chromatography B, volume 848.